Background: Biliary tract cancer (BTC) is an aggressive malignancy with poor prognosis and limited therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated efficacy in tumors with homologous recombination repair (HRR) deficiency. However, actionable BRCA1/2 mutations are rare in BTC. Epigenetic modulation via DNA methyltransferase (DNMT) inhibition is a proposed strategy for inducing an HR-deficient (“BRCAness”) phenotype and thereby enhancing therapeutic response to PARP inhibitors. This study aimed to determine whether the DNMT inhibitor azacitidine (AZA) enhances the antitumor effects of the PARP inhibitor niraparib (NIR) and to identify molecular mechanisms underlying this interaction.Methods: Two BTC cell lines, TFK-1 and RBE, were treated with AZA and/or NIR or subjected to siRNA-mediated DNMT1, DNMT3A, or DNMT3B knockdown. Functional analyses included homologous recombination (HR) assays, flow cytometric evaluation of cell-cycle distribution and apoptosis, proliferation and survival assays, and IC50determination. Whole-transcriptome RNA sequencing was performed to identify differentially expressed genes after AZA treatment or DNMT3B knockdown, followed by validation via qPCR and Western blotting. To explore epigenetic regulation, whole-genome bisulfite sequencing was performed on TFK-1 cells following DNMT3B knockdown.Results: AZA treatment decreased HR frequency in a dose-dependent manner and enhanced the sensitivity of BTC cells to NIR, as evidenced by increased apoptosis, suppressed proliferation, and reduced IC50values. DNMT3B knockdown recapitulated these effects, establishing a causal relationship between DNMT3B suppression and disrupted HR repair. RNA sequencing identified opioid growth factor receptor (OGFR) as a commonly upregulated gene after DNMT3B knockdown. Functional validation showed that OGFR overexpression reduced HR activity, increased apoptosis, and enhanced NIR sensitivity. Contrarily, OGFR knockdown conferred relative resistance. Whole-genome bisulfite sequencing showed no significant CpG methylation changes at the OGFR promoter region, indicating that OGFR induction is mediated through DNMT3B-dependent transcriptional regulation rather than direct promoter demethylation.Conclusions: DNMT3B inhibition sensitizes BTC cells to PARP inhibitors by disrupting HR repair. OGFR was identified as a novel regulator of HR and PARP inhibitor sensitivity, controlled via noncanonical DNMT3B-dependent transcriptional mechanisms that operate independently of CpG methylation. These findings provide new mechanistic insights into the epigenetic control of DNA repair and support the rationale for combining DNMT and PARP inhibitors as a promising therapeutic strategy for BTC beyond genetically HR-deficient cases.
背景：胆道癌（BTC）是一种侵袭性强、预后差且治疗选择有限的恶性肿瘤。聚腺苷二磷酸核糖聚合酶（PARP）抑制剂在同源重组修复（HRR）缺陷的肿瘤中已显示出疗效。然而，BTC中可靶向的BRCA1/2突变较为罕见。通过抑制DNA甲基转移酶（DNMT）进行表观遗传调控，是一种诱导HR缺陷（“BRCAness”表型）从而增强对PARP抑制剂治疗反应的策略。本研究旨在确定DNMT抑制剂阿扎胞苷（AZA）是否能增强PARP抑制剂尼拉帕利（NIR）的抗肿瘤作用，并阐明其相互作用的分子机制。 方法：使用AZA和/或NIR处理两种BTC细胞系（TFK-1和RBE），或通过siRNA介导敲低DNMT1、DNMT3A或DNMT3B。功能分析包括同源重组（HR）测定、流式细胞术评估细胞周期分布和凋亡、增殖和存活测定以及IC50测定。对AZA处理或DNMT3B敲低后的细胞进行全转录组RNA测序以鉴定差异表达基因，并通过qPCR和Western blotting进行验证。为探索表观遗传调控，对DNMT3B敲低后的TFK-1细胞进行了全基因组亚硫酸氢盐测序。 结果：AZA处理以剂量依赖性方式降低了HR频率，并增强了BTC细胞对NIR的敏感性，表现为细胞凋亡增加、增殖受抑制以及IC50值降低。DNMT3B敲低重现了这些效应，证实了DNMT3B抑制与HR修复受损之间的因果关系。RNA测序鉴定出阿片样生长因子受体（OGFR）是DNMT3B敲低后普遍上调的基因。功能验证表明，OGFR过表达降低了HR活性，增加了细胞凋亡，并增强了对NIR的敏感性。相反，OGFR敲低则导致相对耐药。全基因组亚硫酸氢盐测序显示OGFR启动子区域未发生显著的CpG甲基化改变，表明OGFR的诱导是通过DNMT3B依赖的转录调控介导，而非直接的启动子去甲基化。 结论：抑制DNMT3B通过破坏HR修复使BTC细胞对PARP抑制剂敏感。OGFR被鉴定为HR和PARP抑制剂敏感性的新型调控因子，其通过不依赖于CpG甲基化的非经典DNMT3B依赖性转录机制进行调控。这些发现为DNA修复的表观遗传控制提供了新的机制见解，并支持将DNMT抑制剂与PARP抑制剂联合使用作为超越遗传性HR缺陷病例的BTC潜在治疗策略的理论基础。
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